Mutations occurring spontaneously or induced by environmental genotoxicants are a major initiating cause of cancer. Nearly all genotoxicant-induced mutations result from DNA damage and replication of the damaged DNA by specialized translesion synthesis (TLS) DNA polymerases that are less accurate than normal replicative DNA polymerases. The PI's laboratory has recently discovered that TLS DNA polymerase ??(Pol ?) also becomes a powerful source of spontaneous mutations in cells with defects in normal replication machinery. The mechanisms regulating the recruitment of Pol ? in response to replication defects, however, remain obscure. This proposal seeks to further investigate the novel role of Pol ? in mutagenesis in the absence of overt DNA damage. The long-term goal of this study is to define the mechanisms by which genetic and environmental factors regulate the contribution of error-prone DNA polymerases to mutagenesis. The yeast Saccharomyces cerevisiae model system will be utilized in the proposed studies, with the goal of using the data obtained in yeast to further advance our understanding of the mechanisms of mutagenesis in human cells. The Specific Aims of this project are: (1) To define the role of TLS polymerases in the mutagenic response to replication defects; (2) To define the role of the polymerase accessory factor PCNA in the mutagenic response to replication defects; and (3) To determine the effects on the rate of mutation accumulation of a combination of the replication defects with environmental genotoxicant exposure. The proposed work will lead to a better understanding of the mechanisms by which DNA replication defects cause mutations. This information will be important for the development of effective approaches to cancer prevention, particularly in individuals carrying mutations or polymorphisms in DNA replication genes.